Aging is associated with a general decline in immunocompetence, the mechanisms of which are not entirely clear. It is the hypothesis of this proposal that one of the mechanisms leading to immunosenescence is a downregulation of the expression of the CD28 molecule. Optimal responses of CD4 as well as CD8 T cells are dependent on an interaction between the CD28 and CD80/CD86 molecules. It has been shown that the expression of CD28 in elderly individuals declines and a large proportion of CD8 T cells turn CD28 negative. Expression of CD28 is generally more stable in CD4 cells, however, a subset of normal individuals also develop increasing numbers of CD4+ CD28- cells with age. The sponsor's laboratory has shown that the downregulation of CD28 expression correlates with a loss of two transcription factors which bind to sequence motifs in the minimal promoter of the CD28 gene. It is the goal of the proposal to further characterize these two transcription factors and to monitor their expression during senescence. The following specific aims are proposed. Specific aim 1. To isolate and characterize CD28-specific transcription factors. A. To screen a T cell expression library. We propose first to use a Southwestern blot analysis to demonstrate that DNA binding proteins in nuclear extracts from CD28+ cells can be detected by using radiolabelled probes. Positive results would suggest that screening of a T cell cDNA expression library is a feasible approach. We then propose to screen a Jurkat expression library for phage clones containing the appropriate cDNA. B. To use a yeast one-hybrid system to identify the CD28 specific transcription factors. This is an alternative strategy. Reporter gene constructs expressing the appropriate DNA binding motif will be stable transfected into yeast which subsequently will be transformed with T cell cDNA library. C. To directly purify the transcription factor protein(s). This approach may be necessary if the transcription factor(s) consists of more than one subunit necessary for DNA binding. Sufficient CD28-specific transcription factors will be purified for microsequencing. Based on the partial protein sequence specific oligonucleotides will be designed to identify the appropriate cDNA. The same strategies will be employed to isolate both transcription factors (binding at sites A and B of the minimal promoter). Specific aim 2. To characterize the expression patterns of CD28-specific transcription factors. Lymphoid and some non-lymphoid tissues will be screened for the expression of the identified sequences by Northern blotting. Different lymphoid lineages, including CD28+ and CD28- cells, will be included. Specific aim 3. To analyze the influence of aging on the expression of CD28-specific transcription factors. Purified CD4+ and CD8+ T cells from young (younger than 30 years of age), medium age (40-50 years of age) and old individuals (older than 70 years of age) will be compared for the expression of the CD28 transcription factors. In addition, these cells will be cultured to assess the influence of in vitro replication and immununosenescence.